Paxillin-dependent regulation of apical-basal polarity in mammary gland morphogenesis

Abstract

Establishing apical-basal epithelial cell polarity is fundamental for mammary gland duct morphogenesis during mammalian development. While the focal adhesion adapter protein paxillin is a well-characterized regulator of mesenchymal cell adhesion signaling, F-actin cytoskeleton remodeling and single cell migration, its role in epithelial tissue organization and mammary gland morphogenesis in vivo has not been investigated. Here, using a newly developed paxillin conditional knockout mouse model with targeted ablation in the mammary epithelium, in combination with ex vivo threedimensional organoid and acini cultures, we identify new roles for paxillin in the establishment of apical-basal epithelial cell polarity and lumen formation, as well as mammary gland duct diameter and branching. Paxillin is shown to be required for the integrity and apical positioning of the Golgi network, Par complex and the Rab11/MyoVb trafficking machinery. Paxillin depletion also resulted in reduced levels of apical acetylated microtubules, and rescue experiments with the HDAC6 inhibitor tubacin highlight the central role for paxillindependent regulation of HDAC6 activity and associated microtubule acetylation in controlling epithelial cell apical-basal polarity and tissue branching morphogenesis.

Paxillin promotes breast tumor collective cell invasion through maintenance of adherens junction integrity

“…Since paxillin did not significantly affect tumor growth, as shown by ki67 staining (Figure S1, B and C), which is consistent with the study showing that in normal mouse mammary gland the proliferation and apoptosis rate is not altered by paxillin knockout (Xu et al, 2019), we assessed whether distant metastasis is impacted by paxillin expression. H&E staining of lung sections and analysis of the surface of the whole lungs from the PyMT tumor mice revealed that the paxillin KO lungs have fewer lung metastases, as compared to paxillin WT lungs (Figure 1, E-G).…”

“…We have previously reported that paxillin is required for normal mouse mammary gland morphogenesis and development in a mammary epithelial cell-specific MMTV-cre-mediated paxillin conditional knockout mouse (Xu et al, 2019). Herein, this mouse line was bred with the MMTV-PyMT tumor mouse model in order to evaluate paxillin function in breast tumor progression.…”

“…Together, these data suggest that paxillin is required for collective invasion in the primary tumor, and maintenance of cell AJs at a non-steady state at the tumor/stroma boundary. Interestingly, although paxillin does not localize to Adherens junctions (AJs) either in vivo or in cultured epithelial cells (Turner et al, 1991;Xu et al, 2019), the normal mammary gland also needs paxillin to form mature AJs during tissue morphogenesis (Figure S2).…”

“…We have previously reported that paxillin inhibits HDAC6 activity to regulate microtubule (MT) (de)acetylation and control of apical protein recycling in the developing mouse mammary gland (Xu et al, 2019). Additionally, paxillin depletion in mesenchymal cells results in disruption of anterograde vesicle trafficking in an HDAC6-dependent manner (Dubois et al, 2017).…”

“…Herein we utilized a newly developed paxillin conditional knockout mouse (Rashid et al, 2017;Xu et al, 2019), in combination with the MMTV-PyMT breast tumor mouse (Lin et al, 2003) that is comparable to human ER, PR negative, HER2 positive breast cancer, to assess paxillin function in tumor progression. Theses in vivo studies, combined with 3D tumor organoid culture and 2D primary cell culture systems, revealed a previously unappreciated role for paxillin in the regulation of AJ cell-cell adhesion integrity necessary for collective tumor cell migration and metastasis.…”